Apparatus for maintenance and treatment of blood in vitro



Oct. 4, 1966 L. JANKAY 3,276,589

APPARATUS FOR MAINTENANCE AND TREATMENT OF BLOOD IN VITRO Filed Dec. 18,1961 2 $heets$heet 1 GAS SOURCE FIG. I

INVENTOR. LESTER (/v/w) JAN/(4) AGE/VT ATTORNEYS L. JANKAY Oct. 4, 1966"APPARATUS FOR MAINTENANCE AND TREATMENT OF BLOOD IN VITRO Filed Dec. 18.1961 2 Sheets-Sheet 2 FIG.

INVENTOR. LESTER(NM/)JANKAY United States Patent 3,276,589 APPARATUS FORMAINTENANCE AND TREAT- MENT OF BLOOD IN VITRO Lester Jankay, 518 RusticRoad, Santa Monica, Calif.

Filed Dec. 18, 1961, Ser. No. 160,376

4 Claims. (Cl. 210175) The invention described herein may bemanufactured and used by or for the Government of the United States ofAmerica for governmental purposes without the payment of any royaltiesthereon or therefor.

The present invention relates to an apparatus for the maintenance andtreatment of blood in vitro and, more particularly, to such method andapparatus where the given blood culture is continuously circulated,oxygenated, dialysed and fed.

Effective maintenance of blood outside of the body has been along-sought goal for a variety of purposes. In vitro bl-ood studies,reconditioning of human blood affected by disease and extracorporealmaintenance of blood as an aid to surgical procedures are but a few. Themain obstacle to such a goal has been the failure to create an in vitroenvironment for blood which satisfactorily approximates its natural inbody (in vivo) environment. Previously-used techniques have failed toproduce an in vitro environment wherein the blood cells can beeffectively maintained with their normal behavior preserved. In theseprior in vitro environments the behavior of the blood cells differsmarkedly from their normal in body behavior; this fact leads to aplurality of manifest difficulties when such in vitro environments areemployed.

The present invention involves means which effectuate an improved invitro environment for blood which satisfactorily approximates thenatural in vivo environment for such blood. To cite a few of the uses ofsuch an in vitro environment as is produced by this invention: it lendsitself to use as a culture medium for effective cultivation of tissuessuch as cancer tissues and bone marrow, among others; previously usedenvironments for such purposes are generally chemically-defined mediawhich produce an artificial environment thus requiring that suchcultivations be carried on in a foreign environment with all thedifficulties that such imposes. Another illustrative application of thein-vivo-like in vitro environment herein created is that it rendersfeasible the study of radiation effects on human blood without thenecessity of exposing the human body to such radiation.

The invention means employed herein utilizes a simpleconstruction andeasily-portable blood chamber wherein the blood is kept in continuouscirculation to maintain suspension of the blood cells, is continuouslyoxygenated and, through dialytic action, has deleterious factors removedtherefrom and compensating factors added thereto. The apparatus hereinis characterized by controlled temperature and pressure aspects for theblood environment; the temperature maintained is substantially the sameas the blood would have in vivo and the blood is subjected to anoxygenating, circulation-inducing gas which maintains the blood at apressure comparable to that of in vivo body capillary or small arterypressure with systolic and diastolic aspects maintained.

An object of the present invention is means to effectively maintainblood in vitro.

Another object is means to treat blood in vitro.

A further object is means to produce an in vitro environment for bloodwhich successfully approximates the natural in vivo environment of theblood.

Still another object is to provide an apparatus for performing thecombined functions of circulating, oxygenat ing, dialysing and feedingblood culture.

A further object is to provide an apparatus for performing any singleone or any combination of the above-cited functions.

An additional object is the provision of a blood circulating anddialysing chamber.

Another object is the provision of a dialysing chamber wherein theliquid being dialysed is maintained in controlled circulation.

Still another object is the provision of a cheap, simple,readily-portable combined blood circulating and dialysing chamber.

Other objects and many of the attendant advantages of this inventionwill be readily appreciated as the same becomes better understood byreference to the following detailed description when considered inconjunction with the accompanying drawing wherein:

FIG. 1 portrays an illustrative embodiment of the invention wherein theblood chamber is shown in perspective and the balance of the apparatusis generally shown schematically; and

FIG. 2 illustrates another embodiment of the blood chamber employable inthe subject invention.

There is shown in FIG. 1 a bifurcated blood circulatingand-dia-lysingchamber 11 consisting principally of two identical legs 12 and 13interlinked at their bottommost portions by a Y-tube fitting 14.

Leg 12 consists principally of a permeable, normallyflaccid,readily-expandable tubular sac member 16 which is surrounded throughoutmost of its length by an outer, tubular, expandable, impermeable sacmember 17. At its upper and lower ends impermeable sac member 17 isfused or otherwise suitably joined to underlying permeable sac member 16to define a sealed-off compartment whose inner wall is formed bypermeable sac member 16 and whose outer wall is formed by impermeablesac member 17. Permeable sac member 16 extends above and below the areasof union between sac member 16- and sac member 17 to form neck-likeportions 18 and 19. Insertedly positioned in these terminal neck-likeportions 18 and 19 of permeable sac member 16 are a pair of boredcylindrically-shaped access fittings 21 and 22. Annular fasteningmembers 23- and 24, disposed outwardly of respective neck-like portionsL8 and 19, hold these neck-like portions 18 and 19 in tight contact withthe outside of access fittings 21 and 22 thus creating a fluid-sealingclosure between the access fittings 21 and 22 and their companionneck-like portions 18 and 19. Leading into the bore of access fitting 21and fixedly positioned therein is an access tube 26. This access tube 26is adapted to connect to an external gas-bearing line 25 for enabling agaseous mixture (later to be described) to be introduced into the upperportion of permeable sac member 16.

Access to the compartment formed between permeable sac member 16 andimpermeable sac member 17 is provided by conduit tubes 28 and 29 whichare secured to impermeable sac member 17 and pass therethrough. Theseconduit tubes 28 and 29 are closed respectively by removable stoppermembers 31 and 32 affixed to their outer ends.

Left-hand leg 13 is identical in structure with right hand leg 12. t Ithas an inner permeable sac member 33 suitably joined at its upper andlower neck-like portions 34 and 36 to respective cylindrically-shapedbored access fittings 37 and 38 by fastening rnembers 39 and 41. Leg 13has an outer impermeable sac member 42 joined to the permeable sacmember 33, as described for leg 12. This impermeable sac member 42 isprovided with upper and lower conduit tubes 43 and 44 for establishingaccess to the compartment defined between the inner permeable sac member33 and the outer impermeable sac member 42. Removable stopper members 46and 47 affixed to the outer ends of conduit tubes 43 and 44 enableclosing off of the compartment. Leading into the bore of upper accessfitting 37 and aflixed to the access fitting 37 is an access tube 48which is adapted to connect to a second gas-bearing line 49 for enablingentry into the upper portion of permeable sac member 33 of the samegaseous mixture as referred to above with respect to gas-bearing line25.

Interconnecting the bottoms of permeable sac members 16 and 33 so as topermit free flow of liquid from one permeable sac member to the other isthe Y-tube fitting 14 whose upper portions 51 and 52 are connectedrespectively to access fittings 22 and 38 and whose lower portion 53 isclosed by a removable stopper member 54.

Access fittings 21, 22, 37 and 38, access tubes 26 and 48, conduit tubes28, 29, 43 and 44, and the upper portions 51 and 52 and lower portion 53of Y-tube fitting 14 are made of glass or nylon or some other suitablematerial which will introduce no undesirable reaction effects into theblood or wash fluid (e.g., plasma).

A suitable material for the stopper members 31, 32, 43, 44 and 54 is amaterial which goes under the tradename of Tygon which describes aseries of vinyl compounds that are unaffected by oils, water, oxidants,acids, bases and salts.

Dialysing chamber 11 is maintained in an incubator 55 which iscontrolled to keep the fluids in the dialysing chamber 11 at atemperature of 37 C.

Gas bearing lines 25 and 49 which lead to the respective legs 12 an 13of dialysing chamber 11 pass through openings in incubator 55 and on toan automatically-controlled valve 56. From this valve 56 there extends aline 57 to a gas source 58 and another line 59 which vents toatmosphere. Gas source 58 provides a gaseous mixture of 95% air andcarbon dioxide, by volume, which is brought to valve 56 by the line 57.Valve 56 operates to alternately connect the respective lines and 49,leading to the respective legs 12 and 13, to the gas source 58 via line57 or to atmosphere vi a line 59. When one of the gas-bearing lines 25and 49 is conducting gas from gas source 58 to its chamber leg, theother of the gas-bearing lines is vented to atmosphere via valve 56 andline 59. The gas in gas source 58 is under such pressure as to deliverthe gaseous mixture to the chamber legs at a pressure of substantially 4centimeters of water. The periods of alternation are such that once perminute each permeable sac member is subjected to aninflationary-deflationary cycle wherein the 95 air5% CO gas is firstadmitted to the permeable sac member at a pressure of 4 cm. of H 0 andthen the permeable sac member has the gas source 58 blocked from it andthis permeable sac member is vented to atmosphere. During this period ofinflation followed by deflation the other permeable sac member (in theother leg of the chamber 11) undergoes a concurrent period of deflationfollowed by inflation. A satisfactory inflation-deflation cycle for eachpermeable sac member is in the order of a minute.

Eyes 61 and 62 are affixed to fastening members 23 and 39, respectively,to enable the legs 12 and 13 to be suitably supported at desiredlocations within incubator 55, as by hooks 63 and 64, for example.

As a portrayal of the efficiency of the in vitro environment developedherein, rat blood, which is noted for its instability in an in vitroenvironment, has been successfully maintained in vitro by the subjectinvention.

With legs 12 and 13 suspended in substantially vertical position inincubator 55, fresh heparinized rat blood is introduced into either orboth of permeable sac members 16 and 33 via their access tubes. Theintroduced blood will flow back or forth between the permeable sacmembers to bring the blood in each permeable sac member to a commonlevel.

With the blood in the permeable sac members 16 and 33, gas-bearing lines25 and 49 are affixed to their respective access tubes 26 and 48.

The wash fluid, preferably consisting of heated compatible blood plasmaat 37 C., is introduced via conduit tubes 28 and 43 in equal amounts togive substantially the same level of plasma in each leg as of blood.Prior to its introduction into chamber 11 the blood plasma has beenheated to and kept at a temperature of 56 C. for 15-20 minutes and thencooled to the 37 C. temperature at which it is introduced into chamber11. This heating of the blood at 56 C. releases from certain of theblood proteins a dialysable factor which promotes maintenance of theblood cells. With the blood located within per-meable sac members 16 and33 and the interconnecting Y-tube fitting 14, the blood plasma islocated in the outer compartments defined between the respectivepermeable and impermeable sac members of each of legs 12 and 13. Asatisfactory ratio of volume of blood plasma to volume of blood isprovided by the use of at least three times, and, preferably, six timesas much blood plasma as blood. When the blood .is to be maintained overa period of time, the blood plasma should be exchanged, i.e., renewed,periodically every three or four days.

The blood-containing permeable sac members 16 and 33 are made ofcellophane, or some like material, which has such permeability as topermit the desired osmotic exchange between the blood and the wash fluid(plasma). The outer impermeable sac members 17 and 42 are made of amaterial such as vinyl, or the like, Which will not be reactive with thewash fluid at any time during the dialytic process and will successfullycontain and support the same.

With the operative amounts of blood in both permeable sac members and ofthe heated plasma (wash fluid) in both of the surrounding compartmentsand the appropriate stopper members in their closing position, valve 56will alternately introduce the air5% CO mixture into each of permeablesac members 16 and 33 where this gaseous mixture under 4 cm. of H 0pressure is brought into direct contact with the blood culture. As thecolumn of blood in one leg is brought under pressure of this gaseousmixture the column of blood in the other leg is being relieved ofpressure from this gaseous mixture (by the venting via valve 56 and line59). As this application of gaseous pressure (followed by release of thegas) takes place alternately first in one leg and then in the other, theblood culture is moved back and forth between the respective permeablesac members 16 and 33 via Y-tube fitting 14 in response to thispressure. With this inflation-deflation cycle occurring in tandem in therespective legs once per minute the blood is kept in circulation. Thedirect contact between the blood and the gaseous mixture of 95% air5% COcauses the blood to be oxygenated.

By virtue of its contact with the Wash liquid (plasma) via the permeablemembranes offered by the permeable sac members, the blood is dialysed tohave added to it nutrients and any other selected compensating factorsand to have subtracted from it into the wash liquid any deleteriousfactors. The induced circulation employed here effects oxygenation ofthe blood, furthers the dialysis process and in its own right obviatesthe deterioration effects associated with stationary cultures. Althoughdiluents other than plasma may be utilized, plasma has been found to bepreferable. The use of heated plasma at 37 centigrade as opposed tonon-heated pasma, yields a minimum of hemolysis and essentially nochange in the hematocrit value from the normal, thus materiallycontributing to the stability and viability of the blood beingprocessed.

It will be noted that, though each leg of the chamber has its permeablesac member alternately undergoing inflation and deflation periods, thecycles in each of the legs 12 and 13 complement each other so that thegaseous mixture is practically in continuous contact at all times withthe blood culture.

The entire process is performed at a controlled temperature of 37 C. bysuitable control of the incubator 55 sq that both the plasma and theblood aremaintained at this temperature. The reason for the temperatureof 37 C. is because this is the temperature of the blood in its natural,in-body environment. A slight variation of this temperature ispermissible, actual body temperature also being susceptible to slightvariation.

The 4 cm. H O pressure for the gas can be varied somewhat. The pressurecriterion is that it create a small pressure similar to that existing inthe capillary or small blood vessels in the body.

FIG. 2 portrays another embodiment of the invention employing a modifiedform of blood circulating-and-dialysing chamber 66. This chambercomprises essentially a wash fluid bag 67, of vinyl or some likeappropriate material, and a substantially U-shaped readily-expandabletubular sac member 68 which is substantially disposed within the bag 67and extends therefrom at its terminal ends. Sac member 68 is composed ofcellophane, or like material, and like the sac members 16 and 33 of theFIG. 1 embodiment, serves both to hold the blood culture and as thedialysing membrane. Bag 67, which is adapted to hold the heated bloodplasma, is of unitary construction and bifurcates at its upper end toform a forward portion 69 and an after portion 71.

Lines 72 as seen in the forward portion 69 represent upper limits of theinterior portion of the bag 67 in such forward portion 69 and are linesof fusion of opposing layers of the vinyl material. The bag 67 issimilarly formed in the after portion 71. This forward portion has beencut along the lines 73 and 75 to form left and right handed flapappendages 74 and 76, respectively, and a central throat portion 77. Oneupper terminal of the U- shaped tubular sac member 68 projects out ofbag 67 via the throat portion 77. Disposed in this projecting terminalof sac member 68 is a bored cylindrically-shaped access fitting 78 inwhich is fixedly positioned an access tube 79 which extends above andbelow the access fitting 78. By means of resilient annular fasteningmember 81, disposed outwardly of throat portion 77 and resilientlybearing against access fitting 78, bag throat portion 77 and theintermediate adjacent terminal end of sac member 68 are maintained intight contact with the external surface of access fitting '78 to createa fluid closure of bag 67 in the region of throat portion 77 and a likefluid closure of the projecting terminal end of sac member 68, leavingaccess available to this terminal end of sac member 68 only via accesstube 79.

In like manner the after portion 71 of bag 67 is formed with a centralthroat portion 82, respective left and right appendages 83 and 84, anaccess fitting 86, an access tube 87 and a resilient fastening member 88whose action, in union with the encompassed access fitting 86, closes01f the other projecting terminal of sac member 68 and the after bagportion throat 77, leaving access to the sac member terminal via accesstube 87.

Each of the four appendages 74, 76, 83 and 84, respectively, is fittedwith a metallic eye 89 by which the chamber 66 can be suitablysupported.

Like the chamber 11 portrayed in FIG. 1, this chamber 66 of FIG. 2likewise will be supported in an incubator such as 55 and the accesstubes 79 and 87 respectively are adapted to connect to such gas-bearinglines as 25 and 49 when the chamber 66 is in use.

Access to bag 66 is available through conduit tube 91 aflixed to andpenetrating bag 66 and closeable by stopper member 92.

The mode of operation involved with chamber 66 corresponds exactly withthat defined above for chamber 11.

Still another embodiment of the blood'wash fluid chamber would employ awash fluid bag provided with a single throat opening therein and arectilinearly-extending permeable sac member disposed therein which issealed off at its lower end and has its upper end open for accessthereto and which extends out of the wash fluid bag via the throatopening thereof for linkage with such a gasbearing line as 25 or 49. Thefluid access structure will be similar to that reflected in the FIGS. 1and 2 embodiments. Such a wash fluid bag-permeable sac membercombination would be fixedly supported on a platform which would becontinually oscillated back and forth between positions of zero andninety degrees to provide the necessary circulation to the blood culturedisposed in the permeable sac member.

Obviously many modifications and variations of the present invention arepossible in the light of the above teachings. It is intended to coverall changes and modifications which may be made without departing fromthe spirit and scope of this invention.

What is claimed is:

1. Apparatus for the maintenance and treatment of blood in vitrocomprising a first permeable normally flaccid readily expandable tubularsac member having access openings at its upper and lower ends and beingadapted to being positioned in a substantially vertical position; afirst tubular expandable impermeable sac member in substantiallyenclosing position around said first permeable sac member, said firstpermeable sac member and said first impermeable sac member being joinedtogether at their respective upper and lower portions to define a firstclosed compartment whose inner wall is defined by said first permeablesac member and whose outer wall is defined by said first impermeable sacmember; operator-closeable first conduit means extending into andaffixed to said first impermeable sac member for furnishing access tosaid first closed compartment; a second permeable normally flaccidreadily expandable tubular sac member having access openings at itsupper and lower ends and being adapted to being positioned in asubstantially vertical position alongside said first permeable sacmember; a second tubular expandable impermeable sac member insubstantially enclosing position around said second permeable sacmember, said second permeable sac member and said second impermeable sacmember being joined together at their respective upper and lowerportions to define a second closed compartment whose inner wall isdefined by said second permeable sac member and whose outer wall isdefined by said second impermeable sac member; operator-closeable secondconduit means extending into and afl'ixed to said second impermeable sacmember for furnishing access to said second closed compartment; fluidtransfer means connected respectively to said first and said secondpermeable sac members at their lower ends so as to define a fluidpassage way between the inner portions of said respective first andsecond permeable sac members; said first and said second permeable sacmembers being adapted to contain blood and said fluid transfer meansbeing adapted to freely pass blood back and forth between said first andsaid second permeable sac means; each of said closed compartments beingadapted to contain a wash liquid suitable for osmotic exchange with theblood contained by the respective permeable sac members, each of saidpermeable sac members serving as a dialysing membrane; inlet-and-outletmeans connected to said respective permeable sac members at their upperportions; a source of oxygenating gas under pressure; gas-bearing tubemeans interconnecting said gas source and each of said respectiveinlet-and-outlet means; automatic valve means disposed in saidgas-bearing tube means; gas venting means connected to said valve means;said valve means functioning to admit said oxygenating gas underpressure from said gas source to one of said permeable sac members viasaid gas bearing tube means and the inlet-and-outlet means of thatparticular permeable sac member and at the same time to vent said otherpermeable sac member to atmosphere via said gas bearing tube means andsaid gas venting means, and then to repeatedly alternate theabove-described process as it relates to each of said permeable sacmembers so that each of said Permeable sac members alternately undergoesinflation with said oxygenating gas and then is relieved of said gaswhich is vented, the resulting inflation-deflation cycles experienced byeach permeable sac member causing both substantially continuousoxygenation of the blood carried by said permeable sac members and saidfluid transfer means and continuous circulation of said blood in saidapparatus.

2. The apparatus of claim 1 wherein said oxygenating gas is composed ofsubstantially 95% by volume of air and 5% by volume of carbon dioxide.

3. A chamber adapted for the maintenance and treatment of blood in vitrocomprising a Wash fluid bag, adapted to hold a wash fluid, said bagbeing bifurcated at its upper portion to form a first upper portion anda second upper portion, each of said upper portions being formed with apair of end appendages and a mediallylocated throat section, saidappendages being adapted to connect to a supporting member forsupporting said Wash fluid bag in an upright position; a substantiallyU-shaped readily-expandable permeable tubular sac member substantiallydisposed in said fluid bag and having its terminal portions extendingthrough the respective throat sections of said wash fluid bag; conduitmeans disposed in the terminal portions of said U-shaped tubular sacmember for permitting flow of fluid therethrough; fastening meansdisposed outwardly of the terminal portions of said tubular sac memberand outwardly of the throat sections of said wash fiuid bag for closingoff said throat portions and for fastening each of the terminal portionsof said U-shaped tubular sac member to its respective conduit means sothat fluid flow into or out of said U-shaped tubular sac member can beeffectuated only through the respective conduit means associated witheach of the terminal portions of said tubular sac member; and oerator-closeable means aflixed to and penetrating said wash fluid bagfor enabling wash fluid to be deposited in or removed from said bag, asdesired.

4. Apparatus for the maintenance and treatment of blood in vitrocomprising;

first and second permeable sac members formed of a material capable ofpermitting a dialytic osmotic exchange between blood and a blood plasmawash fluid, an impermeable sac in substantially enclosing positionaround each of said first and second permeable sac members, means forintroducing said blood into said first and second permeable members,means for introducing said wash fluid into said impermeable sacs, saidfirst and second sac members being tubular and being normally disposedin a substantially vertical position,

passage means intercommunicating the lower ends of said first and secondpermeable members whereby said introduced blood can flow freely from onemember into the other,

a pressurized source of oXygenating gas,

gas conduit means coupling said source to the upper ends of each of saidfirst and second permeable members,

valve means disposed in said conduit means for alternately applying saidgas under pressure to said first and second permeable members and foralternately venting said permeable members whereby said introduced bloodcan be oxygenated and maintained in a constant transfer motion betweensaid permeable members,

incubator means encasing said impermeable and permeable sacs, and

means for supporting said sacs within said incubator in saidsubstantially vertical positions.

References Cited by the Examiner UNITED STATES PATENTS 2,969,150 1/1961Broman 210-321 2,982,416 5/1961 Bell 210-321 3,002,887 10/1961 Zilliken167-74 3,003,918 10/1961 Sanders 167-74 3,052,238 9/1962 Broman 128-2143,064,647 11/ 1962 Earl 128-214 3,110,308 11/1963 Bellamy 128-214 OTHERREFERENCES Rosenak: Proc. Soc. Exp. Biol. Med., vol. 76, pp. 471- 475,1951.

Schechter: Military Medicine, vol. 126, pp. 593-603, 1961.

Skeggs: Proc. Soc. Exp. Biol. Med, vol. 72, pp. 53- 543, 1949.

REUBEN FRIEDMAN, Primary Examiner. JULIAN S. LEVITT, MORRIS O. WOLK,Examiners.

SAMIH ZAHARNA, P. SABATINE,

Assistant Examiners.

4. APPARATUS FOR THE MAINTENANCE AND TREATMENT OF BLOOD IN VITROCOMPRISING; FIRST AND SECOND PERMEABLE SAC MEMBERS FORMED OF A MATERIALCAPABLE OF PERMITING A DIALYTIC OSMOTIC EXCHANGE BETWEEN BLOOD AND ABLOOD PLASMA WASH FLUID, AN IMPERMEABLE SAC IN SUBSTANTIALLY ENCLOSINGPOSITION AROUND EACH OF SAID FIRST AND SECOND PERMEABLE SAC MEMBERS,MEANS FOR INTRODUCING SAID BLOOD INTO SAID FIRST AND SECOND PERMEABLEMEMBERS, MEANS FOR INTRODUCING SAID WASH FLUID INTO SAID IMPERMEABLESACS, SAID FIRST AND SECOND SAC MEMBERS BEING TUBULAR AND BEING NORMALLYDISPOSED IN A SUBSTANTIALLY VERTICALLY POSITION, PASSAGE MEANSINTERCOMMUNICATING THE LOWER ENDS OF SAID FIRST AND SECOND PERMEABLEMEMBERS WHEREBY SAID INTRODUCED BLOOD CAN FLOW FREELY FROM ONE MEMBERINTO THE OTHER, A PRESSURIZED SOURCE OF OXYGENATING GAS, GAS CONDUITMEANS COUPLING SAID SOURCE TO THE UPPER ENDS OF EACH OF SAID FIRST ANDSECOND PERMEABLE MEMBERS,